Oligonucleotides originating from sequences coding for the surface component of PTLV envelope proteins and their uses

Abstract

The invention relates to the use of oligonucleotides from the nucleotide sequences coding for the amino-terminal region of the surface component (SU) of envelope proteins of PTLV viruses in order to perform methods of detecting every PTLV strain or PTLV-related viruses, e.g. for the detection of novel PTLV variants or viruses comprising sequences related to PTLV SUs. The invention also relates to primer pairs which are used to perform said detection methods and the novel PTLV variants thus detected.

1. A method for implementing processes for detecting any strain of PTLV belonging to HTLV-1, HTLV-2, STLV-1, STLV-2, and STLV-3, said processes comprising:
 a) amplifying a target nucleic acid in a biological sample capable of containing PTLVs, with pairs of degenerate 5′ and 3′ oligonucleotides in the 5′ and 3′ orientation originating from the nucleotide sequences coding for the amino-terminal region of the surface component (SU) of the envelope proteins of the viruses of T lymphoma/leukemia in primates, said viruses collected together under the designation PTLV and being designated HTLV in human and STLV in monkey, wherein said amino-terminal region is a protein fragment delimited on the N-terminal side by an amino acid situated between positions 75 to 90 and on the C-terminal side by an amino acid situated between positions 130 to 145 of the envelope protein of the MT-2 strain of HTLV-1 represented by SEQ ID No: 43, or of the envelope protein of the NRA strain of RTLV-2 represented by SEQ ID NO: 45, or of the envelope protein of the strain of STLV-3 represented by SEQ ID NO: 47, or a virus carrying the sequences belonging to the SU of said PTLV, and 
 b) identifying the strain of PTLV contained in the biological sample by determining the nucleotide fragments that have been amplified. 
 
     
     
       2. The method of  claim 1 , wherein said degenerate oligonucleotides comprise approximately 15 to approximately 30 nucleotides originating from the nucleotide sequences coding for protein fragments delimited on the N-terminal side by an amino acid situated between positions 75 to 90, and on the C-terminal side by an amino acid situated between positions 130 to 145 of the envelope protein of the MT-2 strain of HTLV-1 represented by SEQ ID NO: 43, or of the NRA strain of HTLV-2 represented by SEQ ID NO: 45, or of the envelope protein of the strain of STLV-3 represented by SEQ ID NO: 47, said degenerate oligonucleotides comprising a mixture of oligonucleotides originating from sequences coding for a determined region of approximately 5 to 10 amino acids of the envelope proteins of the different strains of PTLV, and differing from each other by substitution of at least one nucleotide by another in a manner such that each oligonucleotide is capable of coding for the abovementioned determined region originating from the protein fragments of the envelope proteins of the different strains of PTLVs. 
     
     
       3. The method of  claim 1 , wherein pairs of degenerate oligonucleotides comprise approximately 15 to approximately 30 nucleotides originating from nucleotide sequences coding for polypeptide fragments of approximately 5 to approximately 10 amino acids originating from protein fragments delimited by the amino acids situated at positions 80 to 145 of the envelope protein of the MT-2 strain of HTLV-1 represented by SEQ ID NO: 43. 
     
     
       4. The method of  claim 1 , wherein the pairs of degenerate oligonucleotides originate from nucleotide sequences coding for the polypeptide
 fragment 83-88 represented by SEQ ID NO: 1 and 
 the polypeptide fragment 140-145 represented by SEQ ID NO: 2, 
 of the envelope protein of the MT-2 strain of HTLV-1. 
 
     
     
       5. The method of  claim 1 , wherein the degenerate oligonucleotides are degenerate oligonucleotides in 5′ orientation originating from the DNA (+) strand coding for a polypeptide selected from the group consisting of:
 a polypeptide fragment 83-88 of the envelope protein of the MT-2 strain of HTLV-1, said oligonucleotides being chosen from those of formula (I): 
 
       
         
           
                 
                 
                 
                 
               
                     
                   TAYBTNTTYCCNCAYTGG 
                   (I) 
                   SEQ ID NO: 5 
                 
             
                
               
            
           
         
       
       in which:
 Y represents C or T, 
 B represents C, G or T, 
 N represents A, C, G or T, provided that when N represents T, B cannot represent T, 
 and 
 a polypeptide fragment 140-145 of the envelope protein of the MT-2 strain of HTLV-1, said oligonucleotides being chosen from those of formula (II): 
 
       
         
           
                 
                 
                 
                 
               
                     
                   AAYTTYACNCARGARGT 
                   (II) 
                   SEQ ID NO: 8 
                 
             
                
               
            
           
         
       
       in which:
 Y represents C or T, 
 R represents A or G, 
 N represents A, C, G or T. 
 
     
     
       6. The method of  claim 4 , wherein the degenerate oligonucleotides are degenerate oligonucleotides in 3′ orientation originating from the DNA (−) strand coding for
 the polypeptide fragment 140-145 of the envelope protein of the MT-2 strain of HTLV-1, and said oligonucleotides are of formula (III): 
 
       
         
           
                 
                 
                 
                 
               
                     
                   NACYTCYTGNGTRAARTT 
                   (III) 
                   SEQ ID NO: 13 
                 
             
                
               
            
           
         
       
       in which:
 Y represents C or T, 
 R represents A or G, 
 N represents A, C, G or T. 
 
     
     
       7. The method of  claim 1 , wherein the pairs of degenerate oligonucleotides are selected from the group consisting of:
 a degenerate 5′ oligonucleotide corresponding to a mixture of 5′ oligonucleotides originating from a same determined nucleotide region, comprising approximately 15 to approximately 30 nucleotides originating from the DNA (+) strand and coding for polypeptide fragments of approximately 5 to approximately 10 amino acids originating from protein fragments delimited on the N-terminal side by an amino acid situated between positions 75 to 90, and on the C-terminal side by an amino acid situated between positions 135 to 150 of the envelope protein of the MT-2 strain of HTLV-1 represented by SEQ ID NO: 43, or of the envelope protein of the NRA strain of HTLV-2 represented by SEQ ID NO: 45, or of the envelope protein of the strain of STLV-3 represented by SEQ ID NO: 47, said 5′ oligonucleotides being different from each other by substitution of at least one nucleotide by another such that each oligonucleotide is capable of coding for said determined region originating from protein fragments of the envelope proteins of different strains of PTLVs, and 
 a degenerate 3′ oligonucleotide corresponding to a mixture of 3′ oligonucleotides originating from a same determined nucleotide region comprising approximately 15 to approximately 30 nucleotides originating from the DNA (−) strand and coding for the polypeptide fragments of approximately 5 to approximately 10 amino acids originating from protein fragments delimited on the N-terminal side by an amino acid situated between positions 125 to 145, and on the C-terminal side by an amino acid situated between positions 130 to 145 of the envelope protein of the MT-2 strain of HTLV-1 represented by SEQ ID No: 43, or of the envelope protein of the NRA strain of HTLV-2 represented by SEQ ID NO: 45, or of the envelope protein of the strain of STLV-3 represented by SEQ ID NO: 47, said 3′ oligonucleotides being different from each other by substitution of at least one nucleotide by another such that each oligonucleotide is capable of coding for said determined region originating from the protein fragments of the envelope proteins of the different strains of PTLVs, 
 wherein said 5′ oligonucleotides and 3′ oligonucletides are not complementary to each other. 
 
     
     
       8. The method of  claim 6 , wherein the degenerate  5 ′ oligonucleotides are selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 8 and SEQ ID NO: 13. 
     
     
       9. The method of  claim 6 , wherein the degenerate oligonucleotides encode for the protein fragments comprising a sequence delimited on the N-terminal side by the amino acid situated in position 89, and on the C-terminal side by the amino acid situated in position 139 of the envelope protein of the MT-2 strain of HTLV-1 represented by SEQ ID NO: 43, or a sequence comprising an envelope protein of a strain of PTLV other than HTLV-1 wherein the sequence is delimited on the N-terminal side by the amino acid situated in position 85, and on the C-terminal side by the amino acid situated in position 135 of the envelope protein of the NRA strain of HTLV-2 represented by SEQ ID NO: 45, or the sequence is delimited on the N-terminal side by the amino acid situated in position 88, and on the C-terminal side by the amino acid situated in position 144 of the envelope protein of the strain of STLV-3 represented by SEQ ID NO: 47. 
     
     
       10. The method of  claim 9 , wherein the degenerate 5′ oligonucleotides is SEQ ID NO: 5 or SEQ ID NO: 13. 
     
     
       11. A process for detecting any strain of PTLV, belonging to HTLV-1, HTLV-2, STLV-1, STLV-2, and STLV-3, said process comprising:
 contacting a pair of degenerate 5′ and 3′ oligonucleotides as defined in  claim 1 , with genomic DNA or complementary DNA derived from a biological sample containing PTLV, 
 amplifying DNA fragments of said genomic DNA or complementary DNA coding for a fragment of the envelope proteins of the different strains of PTLVs, and 
 detecting the amplified DNA fragments to determine whether said sample contains said PTLV. 
 
     
     
       12. The process for detecting any strain of PTLV belonging to HTLV-1, HTLV-2, STLV-1, STLV-2, and STLV-3 according to  claim 11 , wherein amplifying the DNA fragments comprises two amplification reactions, the second amplification reaction being carried out on a sample of products obtained from the first amplification reaction, the second reaction using the same 5′ oligonucleotides as in the the first reaction and nested 3′ oligonucleotides which are different from the 3′ oligonucleotides used in the first reaction, the nested 3′ oligonucleotides hybridizing with a region of the DNA fragment situated more upstream than the 3′ primers used in the first reaction. 
     
     
       13. The process for detecting any strain of PTLV belonging to HTLV-1, HTLV-2, STLV-1, STLV-2, and STLV-3 according to  claim 12 , the process comprising:
 carrying out a first DNA amplification reaction using degenerate oligonucleotides chosen from the following pairs: 
 (i) oligonucleotides of formula (I)/oligonucleotides of formula (IV), or 
 (ii) oligonucleotides of formula (I)/oligonucleotides of formula (V), or 
 (iii) oligonucleotides of formula (II)/oligonucleotides of formula (V); 
 and carrying out a second DNA amplification reaction on a sample of said first amplification reaction using degenerate oligonucleotides chosen respectively from the following pairs: 
 (i) oligonucleotides of formula (I)/oligonucleotides of formula (III), or 
 (ii) oligonucleotides of formula (I)/oligonucleotides of formula (III or IV), or 
 (iii) oligonucleotides of formula (II)/oligonucleotides of formula (IV), 
 detecting the amplified DNA fragments to determine whether said sample contains said PTLV. 
 
     
     
       14. The process for detecting any strain of PTLV belonging to HTLV-1, HTLV-2, STLV-1, STLV-2, and STLV-3 according to  claim 11 , the process comprising:
 (i) carrying out a first gene amplification reaction using degenerate oligonucleotides chosen in such a way that: 
 the degenerate 5′ oligonucleotides are those of following formula (I): 
 
       
         
           
                 
                 
                 
                 
               
                     
                   PTLVE5′83b 
                   TAYBTNTTYCCNCATTGG 
                   SEQ ID NO: 5 
                 
             
                
               
            
           
         
         Y, B and N being as defined in  claim 5 , or the degenerate 3′ oligonucleotides are those of following formula (V): 
       
       
         
           
                 
                 
                 
                 
               
                     
                   PTLVE3′241b 
                   RTANARNACGTGCCA 
                   SEQ ID NO: 29 
                 
             
                
               
            
           
         
         R, and N being as defined in  claim 6 , 
         (ii) carrying out a second gene amplification reaction using degenerate oligonucleotides chosen in such a way that: 
         the degenerate 5′ oligonucleotides are those of following formula (I): 
       
       
         
           
                 
                 
                 
                 
               
                     
                   PTLVE5′83b 
                   TAYBTNTTYCCNCATTGG 
                   SEQ ID NO: 5 
                 
             
                
               
            
           
         
       
       in which:
 Y represents C or T, 
 R represents A or G, 
 N represents A, C, G or T, or 
 the degenerate 3′ oligonucleotides are those of following formula (III): 
 
       
         
           
                 
                 
                 
                 
               
                     
                   PTLVE3′145a 
                   NACYTCYTGNGTAAAATT 
                   SEQ ID NO: 14 
                 
             
                
               
            
           
         
       
       in which
 Y represents C or T, 
 R represents A or G, 
 N represents A, C, G or T. 
 
     
     
       15. The process for detecting any strains of PTLV belonging to HTLV-1, HTLV-2, STLV-1, STLV-2, and STLV-3 according to  claim 11 , wherein said process is utilized for:
 the diagnosis of pathologies linked to an infection by a PTLV, or by a virus comprising the sequences belonging to the SU of PTLVs, in man or animals, 
 the screening and identification of novel infectious agents in man or animals, 
 the screening of genes with a predisposition or a resistance to the pathologies in man or animals linked to the presence of PTLVs or of related sequences, or to an infection by a PTLV, 
 the screening or the design of novel therapeutic agents comprising the entire or partial sequences of the envelope proteins of novel variants of PTLV thus detected, 
 the screening or the design of novel cell therapy vectors using the tropism proprieties of the entire or partial sequences of the envelope proteins of novel variants of PTLV thus detected. 
 
     
     
       16. The process according to  claim 11 , wherein the biological sample is blood cells, bone marrow, or biopsy. 
     
     
       17. The method of  claim 9 , wherein the degenerate 5′ oligonucleotides are SEQ ID NO: 5 or SEQ ID NO: 14.